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Neuroscience

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Published: December 19th, 2012

DOI:

10.3791/50049

1Department of Radiation Oncology, University of Alabama-Birmingham, 2Department of Radiation Oncology, The Ohio State University Medical School, 3Department of Cell Biology, and Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.

The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

1. Cell Culture

  1. Culture neuronal cells and treat them as needed.
  2. Harvest cells into 15 ml tubes: aspirate media, rinse with phosphate buffered saline (PBS, calcium and magnesium free), add trypsin, collect in 15 ml tubes and neutralize trypsin with appropriate serum containing media.
  3. Spin at 1,000 x g for 5 min.
  4. Aspirate media.
  5. Resuspend cells in PBS.
  6. Spin at 1,000 x g for 5 min.
  7. Aspirate media and resuspend cells in fresh PBS.
  8. .......

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The comet assay has the unique capacity of analyzing individual cells. This is advantageous in identification of subpopulations of cells that demonstrate differential response to cytotoxic agents. A few practical limitations have to be taken into account. The number of cells that can be evaluated individually may vary depending on the individual. The sample size needs to be increased if there is variance in DNA damage within a population. Viable single-cell suspension is critical for this assay since predominant presence.......

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This work was supported by the IMPACT Award from the Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, the Fighting Children's Cancer Foundation, and the Gabrielle's Angel Foundation (to ESY.).

....

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Name Company Catalog Number Comments
Name of the reagent Company Catalog number Comments (optional)
1.5 ml tubes Santa Cruz Biotechnology, Inc Sc-200271
10X TBE (Tris base, boric acid, EDTA) Fisher Scientific BP13331
15 ml tube Fisher Scientific 0553851
Agarose Sigma A5093
Aluminum foil Fisher Scientific 01213101
Beaker Fisher Scientific FB102300
Centrifuge Thermo Scientific 75004261PR
Comet Assay software Comet Assay IV Image Analysis System
Cylinder Fisher Scientific 08555F
EDTA GIBCO 15575
Electrophoresis chamber Thermo Scientific 09528101
Ethanol Fisher Scientific A407P4
Fluorescent microscope Zeiss Axio Vision Any fluorescent microscope with green filter will suffice
Hemacytometer Fisher Scientific 0267152
Low melting point agarose Promega V2111
Microwave Sears
Phosphate buffered saline, calcium free, magnesium free HyClone SH3025601
Power supply BioRad 1645050
Refrigerator Sears
Ruler Staples
Slide Fisher Scientific 12550143
Sodium chloride Sigma S7653
Sodium hydroxide Sigma S5881
Sodium lauryl sarcosinate Fisher Scientific S529
Sybr green Invitrogen S7585
Tray Fisher Scientific 15242B
Tris Base Sigma T6066
Triton-X 100 Sigma T8787
Trypsin HyClone SH3023601
Vortex Fisher Scientific 02216108
Water bath Fisher Scientific 154622Q

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